EP2003198A2 - Peptide for inhibition of influenza infection, inhibitor of influenza infection, liposome, and prophylactic/therapeutic agent for influenza - Google Patents

Peptide for inhibition of influenza infection, inhibitor of influenza infection, liposome, and prophylactic/therapeutic agent for influenza Download PDF

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Publication number
EP2003198A2
EP2003198A2 EP07737966A EP07737966A EP2003198A2 EP 2003198 A2 EP2003198 A2 EP 2003198A2 EP 07737966 A EP07737966 A EP 07737966A EP 07737966 A EP07737966 A EP 07737966A EP 2003198 A2 EP2003198 A2 EP 2003198A2
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Prior art keywords
peptide
influenza virus
alanine
glycine
hemagglutinin
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German (de)
English (en)
French (fr)
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Toshinori Sato
Teruhiko Matsubara
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Keio University
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Keio University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to influenza infection-inhibiting peptides which inhibit influenza virus infection; influenza virus-infection inhibitors which inhibit influenza virus infection; liposomes containing an influenza infection-inhibiting peptide; and influenza preventive/therapeutic agents containing an influenza virus infection-inhibiting peptide.
  • Influenza viruses have two kinds of glycoproteins in their envelope membranes: hemagglutinin (HA) and sialidase (neuraminidase), each of which plays an important role in an establishment of viral infection and a viral budding from a host cell, respectively.
  • Hemagglutinin recognizes a sialic acid-containing sugar chain generally present on the cell membrane of a host (a human or an animal other than a human, such as a mammal, a bird, a reptile, a fish, an amphibian, etc.) as a receptor to which it specifically binds, leading to intracellular endocytosis of the influenza virus.
  • neuraminidase a receptor-destroying enzyme, serves to cleave a sialic acid residue on the host cell membrane or virus' own membrane when the viral particle buds or is released from the host cell.
  • Hemagglutinin involved in the first step of influenza virus infection, has various subtypes based on the diversity of amino acid sequences of the antigen-determining regions (A-E) which are highly mutatable. Since amino acid sequence homology among hemagglutinin subtypes is 25 to 75%, it is extremely difficult to develop influenza vaccines based on antigenicity. On the other hand, the so-called receptor binding pocket region which binds to a receptor of a host cell is comparatively less mutatable and its three-dimensional structure is well conserved ( Y. Suzuki, Prog. Lipid. Res., 33, 429 (1994 ). Therefore, to prevent influenza virus infection, if the function of hemagglutinin, contributing to establishment of infection, is inhibited by specifically binding to it, broad effect will be expected in prevention of influenza virus infection, and thus development of such an agent has been demanded.
  • A-E antigen-determining regions
  • hemagglutinin recognizes and binds to a sialic acid-containing sugar chain of a host receptor
  • a variety of hemagglutinin-binding sugar analogs have been obtained to date ( R. Roy, et al., J. Chem. Soc., Chem. Commun., 1869 (1993 ); M. Mammen, et al., J. Med. Chem., 38, 4179 (1995 ); T. Sato, et al., Chem. Lett., 145 (1999 ); M. Itzstein, et al., Nature, 363, 418 (1993 )).
  • receptor binding pocket regions are comparatively less mutatable and there three-dimensional structure is well conserved, these regions are specific to subtypes of hemagglutinin and their binding constants are not high. For that reason, development of agents acting on influenza viruses in general, regardless of the subtype, has been awaited.
  • 15-residue oligopeptides which prevent influenza virus infection by binding to hemagglutinin were screened for, and 11 types ( WO00/59932 ) and 3 types (Japanese Laid-Open Application No .2002-284798 ; T.Sato, et al., Peptide Science 2001, 329 (2002 )) of oligopeptides were identified ( WO00/59932 ).
  • the existence of a peptides having a higher affinity for hemagglutinin than these oligopeptides was suspected.
  • the present invention has been made in order to provide, by screening for a larger number peptides, a peptide having high affinity for hemagglutinin or a peptide having high inhibitory activity against influenza virus infection, and further to provide a pharmaceutical composition containing such a peptide having a high affinity for hemagglutinin or a peptide having a high inhibitory activity against influenza virus infection.
  • the peptide according to the present invention is a peptide having 4 to 14 residues, including the amino acid sequence of X 1 X 2 X 3 X 4 , wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; and X 4 is proline, alanine, or glycine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 4 to 14 residues, including the amino acid sequence of X 1 X 2 X 3 X 4 as a part of SEQ ID NO: 3, wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; and X 4 is proline, alanine, or glycine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 5 to 14 residues, including the amino acid sequence of X 1 X 2 X 3 X 4 X 5 , wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; and X 5 is arginine or lysine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 5 to 14 residues, including the amino acid sequence of X 1 X 2 X 3 X 4 X 5 as a part of SEQ ID NO: 3, wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; and X 5 is arginine or lysine. It is preferred that X 4 is proline
  • the peptide according to the present invention is the peptide of 5 to 14 residues, including the amino acid sequence of X 1 X 2 X 3 X 4 X 5 as a part of the sequence of X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 HX 9 X 10 X 11 X 12 X 13 X 14 , wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; X 5 is arginine or lysine; X 6 is serine or threonine; X 7 is cysteine or methionine; X 8 is alanine, glycine, isoleucine, valine, or leucine; X
  • the peptide according to the present invention has the amino acid sequence of any one of SEQ ID NOs: 38, 42 to 44, and 52.
  • the peptide according to the present invention has an amino acid sequence of any one of SEQ ID NOs: 38, 42 to 44, and 52 from/to which one or several amino acid residues are substituted, deleted, or added, and has inhibitory activity against influenza virus infection.
  • the aforementioned peptide is alkylated.
  • the peptide according to the present invention is a peptide having 4 to 14 residues, including the amino acid sequence of X 4 X 3 X 2 X 1 , wherein X 4 is proline, alanine, or glycine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; and X 1 is alanine, glycine, isoleucine, valine, or leucine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 5 to 14 residues, including the amino acid sequence of X 5 X 4 X 3 X 2 X 1 , wherein X 5 is arginine or a lysine, X 4 is proline, alanine, or glycine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; and X 1 is alanine, glycine, isoleucine, valine, or leucine. It is preferred that X 4 is proline.
  • the peptide according to the present invention has the amino acid sequence of SEQ ID NO: 56 or 57.
  • the peptide according to the present invention has an amino acid sequence of either SEQ ID NO: 56 or 57 from/to which one or several amino acid residues are substituted, deleted, or added, and has inhibitory activity against influenza virus infection.
  • each of the aforementioned peptide is alkylated.
  • influenza virus infection-inhibitor contains the peptide of any one of the aforementioned peptides as an active ingredient.
  • This influenza virus-infection inhibitor inhibits infections of both an influenza virus having hemagglutinin H1 and an influenza virus having hemagglutinin H3.
  • influenza preventive/therapeutic agent contains the peptide of any one of the aforementioned peptides as an active ingredient. This influenza preventive/therapeutic agent is effective for infections of both an influenza virus having hemagglutinin H1 and an influenza virus having hemagglutinin H3.
  • the DNA according to the present invention encodes any one of the aforementioned peptides.
  • the expression vector according to the present invention includes the aforementioned DNA.
  • the cell according to the present invention is introduced with the aforementioned expression vector and secretes any one of the aforementioned peptides.
  • the liposome according to the present invention contains the peptide of any one of the aforementioned peptides.
  • the peptide in this liposome is preferably alkylated.
  • the influenza virus infection inhibitor according to the present invention is an influenza virus infection inhibitor containing any one of the aforementioned peptides, in which the peptide is modified so as to have amphiphilicity and has formed a peptide aggregate.
  • the modification in the influenza virus infection inhibitor is preferably alkylation.
  • the peptide aggregate according to the present invention refers to the aforementioned peptide aggregate.
  • the peptide according to the present invention has the amino acid sequence of X 2 X 3 X 4 X 5 X 6 X 7 X 8 (SEQ ID NO 49:s2(2-8) (general formula)), wherein X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; X 5 is arginine or lysine; X 6 is serine or threonine; X 7 is cysteine or methionine; and X 8 is alanine, glycine, isoleucine, valine, or leucine. It is preferred that X 4 is proline.
  • the peptide according to the present invention has an amino acid sequence of X 2 X 3 X 4 X 5 X 6 X 7 X 8 (SEQ ID No: 49) from/to which one or several amino acid residues are substituted, deleted, or added, and has inhibitory activity against influenza virus infection. It is preferred that X 4 is proline.
  • the aforementioned peptide is alkylated.
  • influenza virus infection inhibitor contains any one of the aforementioned peptides as an active ingredient. It should be noted that this influenza virus infection inhibitor inhibits infection of an influenza virus having hemagglutinin H1.
  • FIG. 1 is a graph comparing the binding activity of A-1 to hemagglutinin with that of H3G-1 using ELISA in an example according to the present invention.
  • FIG.2 is a graph comparing the binding activities of peptides having the amino acid sequence of SEQ ID NOs : 2 to 18 to hemagglutinin with that of A-1 (SEQ ID NO: 1) using ELISA in an example according to the present invention.
  • FIG.3 (A) is a figure showing the relationship between time (x-axis) and the resonance (y-axis) of s-2 peptide at each concentration in an example according to the present invention.
  • FIG. 4 (A) is a graph showing the changes of the resonance (y-axis) relative to time (x-axis) at a peptide concentration of 100 ⁇ M of each hemagglutinin-binding peptide.
  • (B) shows the relative value of the hemagglutinin-binding peptide when the amount of binding of A-1 peptide was taken as 1 at a peptide concentration of 200 ⁇ M of each hemagglutinin-binding peptide in an example according to the present invention.
  • Table 1 shows the amino acid sequences of the hemagglutinin-binding peptides as used herein.
  • peptide A-1 having the amino acid sequence of SEQ ID NO:1 has the highest binding activity among the hemagglutinin-binding peptides identified to date (see WO00/59932 and the Specification and Examples of Japanese Laid-Open Application No. 2002-284798 )
  • the polypeptides having the amino acid sequences of SEQ ID NOs : 2 to 7, 9 to 10, and 12 to 18 have even higher binding activity to hemagglutinin than A-1, as shown in the Examples.
  • these polypeptides having the amino acid sequences of SEQ ID NOs: 2 to 7, 9 to 10, and 12 to 18 bind to both of hemagglutinins H1 and H3.
  • hemagglutinin-binding peptides according to the present invention may be a peptide having an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added, from/to SEQ ID NOs: 2 to 7, 9 to 10, and 12 to 18, and has a high inhibitory activity against hemagglutinin.
  • hemagglutinin-binding peptide to hemagglutinin
  • ELISA hemagglutinin-binding peptide
  • any other technique such as RIA, EIA, Western blotting, or the like, as long as it is the technique enabling quantitative measurement of protein-protein interaction.
  • Table 2 shows the amino acid sequences of the influenza virus infection-inhibiting peptides as used herein.
  • Table 2 No. a. a. seq. Residues Seq. No. 3 ARLPRTMVHPKPAQP 15 Seq. No. 38 ARLPRTMV 8 Seq. No. 42 ARLPRTM 7 Seq. No. 43 ARLPRT 6 Seq. No. 44 ARLPR 5 Seq. No. 52 ARLP 4 Seq. No. 56 RPLRA 5 Seq. No. 57 PLRA 4
  • influenza virus infection-inhibiting peptides having the amino acid sequences of SEQ ID NOs: 38, 42 to 44, 52, 56, and 57 can strongly inhibit influenza virus infection into host cells, as described in the following Examples.
  • influenza virus infection-inhibiting peptides may be peptides having an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added, from/to SEQ ID NOs : 38, 42 to 44, 52, 56, and 57 and has inhibitory activity against influenza virus infection.
  • the inhibition of influenza virus infection into host cells by the influenza virus infection-inhibiting peptide can be measured by, for example, the plaque assay method.
  • a peptide having the ARLP (SEQ ID NO: 52) sequence with, for example, a substitution of A by an amino acid having an aliphatic side chain (for example, G, V, L, or I), a substitution of R by an amino acid having a basic side chain (for example, K), and/or a substitution of L by an amino acid having aliphatic side chain (for example, G, V, A, or I), is considered to have similar activity as the peptide having the ARLP (SEQ ID NO 52) sequence.
  • Such sequences can be represented by the general formula of X 1 X 2 X 3 X 4 (SEQ ID NO:58:s2 (1-4) (general formula)), wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; and X 4 is proline, alanine, or glycine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 4 to 14 residues, which has the aforementioned amino acid sequence (SEQ ID NO: 58).
  • a peptide having the ARLPR (SEQ ID NO: 44) sequence with, for example, a substitution of A by an amino acid having an aliphatic side chain (for example, G, V, L, or I), substitution of R by an amino acid having a basic side chain (for example, K), and/or a substitution of L by an amino acid having aliphatic side chain (for example, G, V, A, or I) is considered to have similar activity as the peptide having the ARLPR (SEQ ID NO: 44) sequence.
  • a peptide with a substitution of P by A or G is considered to have similar activity as the peptide having the ARLPR (SEQ ID NO: 44) sequence.
  • Such sequences can be represented by the general formula of X 1 X 2 X 3 X 4 X 5 (SEQ ID NO 48:s2 (1-5) (general formula)), wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; and X 5 is arginine or lysine. It is preferred that X 4 is proline.
  • the peptide according to the present invention is a peptide having 5 to 14 residues, which has the aforementioned amino acid sequence (SEQ ID NO: 48).
  • amino acid sequence of SEQ ID NO: 3 can be represented by the general formula of X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 HX 9 X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 50:s2 (general formula)), wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; X 5 is arginine or lysine; X 6 is serine or threonine; X 7 is cysteine or methionine; X 8 is alanine, glycine, isoleucine, valine, or leucine; X 9 is proline, alanine, or glycine;
  • the peptide according to the present invention may be a peptide having 4 to 14 residues containing the amino acid sequence (SEQ ID NO 58) as a part of the above-mentioned amino acid sequence (SEQ ID NO 50), and preferably a peptide having 4 to 14 residues containing this amino acid sequence (SEQ ID NO: 58) as a part of the SEQ ID NO: 3.
  • the peptide according to the present invention may be a peptide having 5 to 14 residues containing the amino acid sequence (SEQ ID NO: 48), and preferably a peptide having 4 to 14 residues containing the amino acid sequence (SEQ ID NO: 48).
  • the peptide according to the present invention may be a peptide having an inverted sequence of the ARLPR (SEQ ID NO: 44) sequence or the ARLP (SEQ ID NO: 52) sequence.
  • the RPLRA (SEQ ID NO: 56) sequence which is a inverted sequence of the ARLPR (SEQ ID NO: 44) sequence
  • the PLRA (SEQ ID NO: 57) sequence which is a inverted sequence of the ARLP (SEQ ID NO 52) sequence
  • substitution of A by an amino acid having an aliphatic side chain for example, G, V, L, or I
  • a substitution of R by an amino acid having a basic side chain for example, K
  • a substitution of L by an amino acid having aliphatic side chain for example, G, V, A, or I
  • a peptide with a substitution of P by A or G is considered to have similar activity as the peptide having the RPLRA (SEQ ID NO: 56) sequence or the PLRA (SEQ ID NO: 57) sequence.
  • sequences can be represented by the general formula of X 5 X 4 X 3 X 2 X 1 (SEQ ID NO 59:s2 (r1-5) (general formula)) or X 4 X 3 X 2 X 1 (SEQ ID NO 60:s2 (r1-4) (general formula)), wherein X 1 is alanine, glycine, isoleucine, valine, or leucine; X 2 is arginine or lysine; X 3 is alanine, glycine, isoleucine, valine, or leucine; X 4 is proline, alanine, or glycine; and X 5 is arginine or lysine. It is preferred that X 4 is proline.
  • hemagglutinin on the influenza virus specifically binds to a receptor of the host cell and enters into the cell by using the receptor as a scaffold.
  • the hemagglutinin-binding peptide according to the present invention specifically binds to the hemagglutinin. Due to this binding, hemagglutinin is prevented from binding to a host cell receptor, and therefore, infection of the influenza virus to the host cell can be inhibited.
  • the hemagglutinin-binding peptide according to the present invention can inhibit infection of an influenza virus to a host cell. Further, the influenza virus infection-inhibiting peptide according to the present invention was able to inhibit infection of an influenza virus to a host, as shown in the following Examples. Therefore, the hemagglutinin-binding peptide and the influenza virus infection-inhibiting peptide according to the present invention can suppress influenza virus proliferation in vivo by being administered to a human or a vertebrate other than a human afflicted with influenza.
  • the hemagglutinin-binding peptide and the influenza virus infection-inhibiting peptide can also prevent infection of an influenza virus by being administered in advance to the body of a human or a vertebrate other than a human even after the influenza virus has entered the body.
  • the hemagglutinin-binding peptide according to the present invention can also be used as a tool for elucidating hemagglutinin-mediated influenza virus infection and the involved various cell functions and biological phenomena.
  • influenza virus targeted by the present invention is not particularly limited in types or origins, as long as it has at least either hemagglutinin H1 or hemagglutinin H3, and it may be any one of type A, type B or type C, human isolates, other mammalian isolates such as swine isolates and equine isolates, or avian isolates.
  • the pharmaceutical compositions containing the hemagglutinin-binding peptide according to the present invention or the influenza virus infection-inhibiting peptide as an active ingredient may include influenza virus infection inhibitors for inhibiting infection of influenza viruses to host cells, as well as influenza therapeutic agents for treating patients afflicted with influenza, and influenza preventive agents to be administered preventively before the patient have been afflicted with influenza.
  • compositions can be made as preparations containing as an active ingredient the hemagglutinin-binding peptide or the influenza virus infection-inhibiting peptide, and a pharmaceutically acceptable carrier as necessary, and then administered to influenza virus-infected humans or vertebrates other than humans or administered preventively to uninfected humans or vertebrates other than humans.
  • the pharmaceutically acceptable carrier to be used herein can be appropriately selected from among the conventional carriers depending on the form of the pharmaceutical composition to be prepared.
  • the pharmaceutical composition when the pharmaceutical composition is prepared in the form of an aqueous solution, purified water (sterile water), a physiological buffer solution, glycol, glycerol, or an injectable organic ester, such as olive oil, can be used as the carrier.
  • the composition can contain a conventionally used stabilizer, recipient, etc.
  • the mode of administering these pharmaceutical compositions is not particularly limited and may be appropriately determined depending on conditions such as formulations, the patient's age, sex, and others , as well as severity of the illness, etc.
  • Preferred formulations include injections, drops, sprays (aerosol), nasal drops, and inhalations.
  • the routs of administration can be either oral or parenteral, and specific examples include oral administration, intravenous administration, intrarterial administration, intramuscular administration, intracutaneous administration, intraperitoneal injection, intratracheal administration, inhalation administration, and sublingual administration.
  • the agent of the present invention is preferably administered as a pharmaceutical composition through the administration route such as oral administration, intratracheal administration, oropharynx administration, and inhalation, etc. to a human influenza virus-infected human or a vertebrate other than human.
  • the agent as a spray, spray aerosol, or an inhalation, it can be administered through the administration route such as oral administration, intratracheal administration, oropharynx administration, and inhalation, thereby making it possible to directly inhibit infection of an influenza virus to respiratory epithelium cells.
  • the daily dose of these pharmaceutical compositions can be appropriately changed depending on the patient's symptom, age, body weight, sex, duration of treatment, therapeutic effect, mode of administration, etc., and is not limited as long as influenza infection can be inhibited with inevitable side effects within an acceptable range.
  • the preparation may be administered as one dose a day, as well as multiple doses a day.
  • compositions may be used alone or in combination with other agents (for example, another antiviral agent, an anti-inflammatory agent, a symptom-relieving agent, etc.).
  • agents for example, another antiviral agent, an anti-inflammatory agent, a symptom-relieving agent, etc.
  • the agent can also be used, when influenza vaccines cannot be used, e.g., on high-risk patients who can potentially develop influenza before the effect of the vaccine is exhibited, on patients in whom a sufficient effect of influenza vaccine is not exhibited because of immunodeficiency or the like, and on patients in whom vaccination is contraindicated.
  • the agent when administered orally to vertebrates other than humans, can be taken by mixing with drinking water or feed.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide according to the present invention can be chemosynthetically produced in accordance with the conventional method: for example, it can be produced by peptide synthesis using the conventional liquid-phase and solid-phase methods. More specifically, peptide synthesis may be performed by using methods such as the stepwise elongation method in which individual amino acids are serially bound one by one based on the amino acid sequence information as described above, thereby elongating the chain; and the fragment condensation method in which fragments each consisting of several amino acids are synthesized in advance and these fragments are subjected to coupling reaction.
  • the condensation to be adopted in th aforementioned peptide synthesis can also be performed by following various known methods. Specific methods can be exemplified by the azide method, mixed acid anhydride method, DCC method, active ester method, redox method, diphenylphosphoryl azide (DPPA) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, or the like) method, Woodward method, etc. Solvents which may be used in these methods can also be appropriately selected from among the common solvents well known to be used in peptide condensation reactions of this kind.
  • solvent examples include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, and mixtures of these solvents.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ethyl acetate examples of these solvents.
  • the carboxyl groups of amino acids or peptides not involved in the reactions can be generally protected by esterification, for example, to form a lower alkyl ester (e.g., methyl ester, ethyl ester, tert-butyl ester, or the like) or an aralkyl ester (e.g. , benzyl ester, p-methoxybenzyl ester, p-nitrobenzyl ester, or the like).
  • a functional group in an amino acid having the functional group as a side chain e.g., Tyr
  • guanidino group of Arg for example, can be protected with a suitable protective group such as a nitro group, a tosyl group, 2-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group, or the like.
  • Deprotection of the aforementioned protected amino acids, peptides, and the peptide finally obtained in the present invention can also be performed by the conventionally used methods, such as catalytic reduction or other method using liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, or the like.
  • the peptide thus prepared can be appropriately purified by the conventional method commonly used in the field of peptide chemistry, such as ion-exchange resin chromatography, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution, or the like.
  • a DNA encoding the aforementioned hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide may be prepared, and incorporated into an expression vector, which is introduced into eukaryotic cell such as a cultured cell, or a prokaryotic cell such as Escherichia coli; and the peptide expressed in such cells may be purified.
  • the expression vector is appropriately selected depending on the host cell to be transfected. Construction of a vector, including incorporation of a DNA encoding the peptide etc., introduction to host cells, expression in host cells, and preparation of extract of host cells, can be performed in accordance with the conventional method using molecular biological approaches.
  • the desired peptide can be purified from the extract by the above-described conventional method commonly used in the field of peptide chemistry.
  • a DNA encoding a peptide constructed by adding a tag e.g., a His tag, a Flag tag, a GST tag, etc.
  • a tag e.g., a His tag, a Flag tag, a GST tag, etc.
  • the expression vector so that the peptide portion can be purified by cleaving the tag.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide may be finally purified.
  • the aforementioned hemagglutinin-binding peptide and influenza virus infection-inhibiting peptide of the invention can be suitably modified.
  • chemical modification such as alkylation, lipidation (phospholipidation), or the like
  • the cell affinity or tissue affinity of hemagglutinin-binding peptide and influenza virus infection-inhibiting peptide can be enhanced and its blood half-life can be extended, whereby its pharmacological effect can be enhanced.
  • Alkylation of the hemagglutinin-binding peptide and influenza virus infection-inhibiting peptide can be performed in accordance with the conventional method.
  • alkylation can be easily performed by an amide bond-forming reaction between a fatty acid and the N-terminal amino group of the hemagglutinin-binding peptide, as with the above-described peptide synthesis).
  • the fatty acid to be used can be widely selected and a straight-chain acid or a branched-chain acid, saturated or unsaturated, may be used, but, particularly preferred is a fatty acid present in the living body.
  • fatty acid examples include fatty acids of about 12 to 20 carbon atoms: saturated fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, etc.; and unsaturated fatty acids such as oleic acid, eraidic acid, rinolic acid, linolenic acid, arachidonic acid, etc.
  • the alkylation can also be performed by an amide bond-forming reaction between an alkylamine and the C-terminal carboxyl group of the hemagglutinin-binding peptide, as with the above-described peptide synthesis.
  • the alkylamine to be used can be selected from among various alkylamines, as with the above-mentioned fatty acids, but particularly preferred is an alkylamine having fatty acid chains (of about 12 to 20 carbon atoms) present in the living body.
  • Lipidation of the hemagglutinin-binding peptide and influenza virus infection-inhibiting peptide can also be performed in accordance with the conventional method (refer to, for example, New Current, 11(3), 15-20 (2000 ); Biochemica et Biophysica Acta., 1128, 44-49 (1992 ); FEBS Letters, 413, 177-180 (1997 ); J. Biol. Chem., 257, 286-288 (1982 ), etc.).
  • a phospholipid can be attached at the 2-hydroxyl group or 3-phosphoric group of the hemagglutinin-binding peptide and influenza virus infection-inhibiting peptide via any spacer by the condensation method.
  • a reactive SH group to be used for the condensation can be introduced in advance by adding a cysteine-containing amino acid sequence of arbitrary length (usually several residues) to the N- or C-terminus of the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide.
  • the phospholipid used in this lipidization is not particularly limited, and, for example, phosphatidic acid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, etc., all of which are composed of the above-mentioned fatty acids, can be used.
  • Such alkylated or lipidized hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide can be used as the lipid component when liposomes are prepared.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide is presented on the liposome, and as a result, the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide can be effectively utilized in a liposome preparation as described hereafter.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide according to the present invention can be prepared as a liposome preparation.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide according to the present invention is retained in a liposome in which either acidic phospholipid(s) alone, or neutral and acidic phospholipids constitute the membrane.
  • Examples of the acidic phospholipids to constitute the membrane include natural or synthetic phosphatidylglycerols (PGs) such as dilauroylphosphatidylglycerol (DLPG) dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG) dioleoylphosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (egg yolk PG), hydrogenated egg yolk phosphatidylglycerol; and natural or synthetic phosphatidylinositols (PIs) such as phosphatidylinositol (DLPI), dimyristoylphosphatidyl inositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (
  • neutral phospholipids examples include natural or synthetic phosphatidylcholines (PCs) such as soybean phosphatidylcholine, egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), distearoylphosphatidylcholine (DSPC), myristoylpalmitoylphosphatidylcholine (MPPC), palmitoylstearoylphosphatidylcholine (PSPC) and dioleoylphosphatidylcholine (DOPC); and natural or synthetic phosphatidylethanolamines (PEs), such as soybean phosphatidylethanolamine, egg yolk phosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, hydrogenated egg yolk phosphati
  • the aforementioned liposome membrane is formed, according to the conventional method by using either the aforementioned acidic phospholipid(s) alone or neutral and acidic phospholipids in combination, as the constituent of the membrane.
  • acidic phospholipid is contained, by proportion, about 0.1 to about 100 mol%, preferably about 1 to about 90 mol%, more preferably about 10 to about 50 mol%, in the liposome membrane constituents.
  • cholesterol etc. can be added when preparing the above-mentioned liposomes.
  • the fluidity of phospholipid can be adjusted so that liposomes can be prepared more simply and conveniently.
  • cholesterol can be added at or up to an equal volume to phospholipid, preferably in a 0.5 to 1-fold volume.
  • the ratio between the number of molecules in a lipidized hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide and that of acidic phospholipid(s) is typically about 1:0.5 to 100, preferably about 1:1 to 60, and more preferably about 1:1.5 to 20.
  • multilayer vesicles are prepared, for example, as follows. First, the lipid is dissolved in an organic solvent (chloroform, ether, etc.), and then put in a round bottom flask. The organic solvent is removed under a nitrogen current or under reduced pressure to form a thin lipid film at the bottom of the flask. Optionally, the flask may be left in a desiccator under reduced pressure for complete removal of residual solvent. Subsequently, the lipid is hydrated by adding an aqueous solution of the lipidized hemagglutinin-binding peptide onto the thin lipid film to yield a turbid, opalescent liposome suspension.
  • organic solvent chloroform, ether, etc.
  • LUVs Large unilamellar vesicles
  • Ca 2+ can be produced by adding Ca 2+ to small unilamellar phosphatidylserine vesicles, which are fused to form a cylindrical sheet, followed by addition of the chelating agent EDTA to remove the Ca 2+ ( Biochim. Biophys. Acta 394, 483-491, 1975 ).
  • LUVs can also be produced by injecting the lipid in ether into an aqueous solution at about 60°C to evaporate the ether ( Biochim. Biophys. Acta 443, 629-634, 1976 ).
  • peptide-incorporated liposomes can be prepared by adding the aqueous solution of a peptide to the prepared liposome suspension.
  • a liposome with small particle-size can be prepared by the French press method ( FEBS lett. 99, 210-214, 1979 ).
  • the freeze-drying method Chem. Pharm. Bull, 32, 2442-2445, 1984
  • the freezing-thawing method Chem. Pharm. Bull. 33, 2916-2923, 1985 ), both of which were reported by Ohsawa et al., may also be adopted.
  • the content of the lipidized hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide contained in a liposome prepared in this manner may be a few to a few tens of mol%, preferably 5 to 20 mol%, but, typically, about 5 mol% of all the lipids.
  • the liposomes thus prepared can be made homogeneous in terms of particle size by dialysis ( J. Pharm. Sci. 71, 806-812, 1982 ) or by a filtrating method using a polycarbonate membrane ( Biochim. Biophys. Acta 557, 9-23, 1979 ; Biochim. Biophys. Acta 601, 559-571, 1980 ).
  • dialysis, a gel filtration method, or a centrifugation method can be adopted (see " Liposome.
  • the liposome dispersion thus prepared may appropriately contain, as a required additive for formulating the preparation, various kinds of known substances, such as antiseptics, isotonizing agents, buffers, stabilizers, solubilizing agents, and absorption accelerators, etc.
  • the liposome dispersion can also be diluted with a solution containing these additives or with water, as required.
  • additives include: preservatives, which are effective for fungi and bacteria, such as benzalkonium chloride, benzethonium chloride, chlorhexidine, parabens (methyl paraben, ethyl paraben, etc.), and thimerosals; isotonizing agents, such as D-mannitol, D-sorbitol, D-xylitol, glycerol, glucose, maltose, sucrose, and electrolytes (e.g., polyhydric alcohols and sodium chloride); and stabilizers, such as tocopherol, burylhydroxyanisole, butylhydroxytoluene, ethylenediaminetetraacetic acid salt (EDTA), and cysteine.
  • preservatives which are effective for fungi and bacteria, such as benzalkonium chloride, benzethonium chloride, chlorhexidine, parabens (methyl paraben, ethyl paraben, etc.), and th
  • a liposome preparation can be manufactured by further encapsulating another agent, for example, an antiviral agent, into the above-mentioned liposome containing the hemagglutinin-binding peptide according to the present invention.
  • another agent for example, an antiviral agent
  • the liposome preparations may be manufactured in accordance with the methods specifically described in the reference by, for example, Woodle et al. (Long Circulating Liposomes: old drug, New therapeutic., M. C. Woodle, G. Storm, Eds: Springer-Verlag, Berlin (1998 )) or Namba et al. (Liposomal applications to cancer therapy, Y. Namba, N. Oku, J. Bioact. Compat. Polymers, 8, 158-177 (1993 )).
  • the content of the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide in a pharmacological compositions including the above-mentioned liposome preparations according to the present invention is not limited and may be appropriately selected; it is typically about 0.0002 to 0.2 (w/v%), preferably about 0.001 to 0.1 (w/v%) in the composition.
  • the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide may be used without treatment in itself as an influenza virus-infection inhibitor, but by forming peptide aggregates, it can inhibit the infection of an influenza virus to a host cell with higher efficiency.
  • the peptide aggregate as described herein refers to micelle (a spherical-shaped structure with the hydrophobic group facing inward and the hydrophilic group positioned outward), or a self-assembled peptide (an aggregate with a diameter of a few hundreds of nanometers to a few micrometers spontaneously assembled in a solution).
  • an amphiphilic compound having both the hydrophilic and hydrophobic character by modifying the hemagglutinin-binding peptide or the influenza virus infection-inhibiting peptide can facilitate the formation of a peptide aggregate.
  • an amphiphilic compound can be prepared by alkylating the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide. Alkylation can be performed as described above.
  • peptide aggregates can be formed by allowing the hemagglutinin-binding peptide or the influenza virus infection-inhibiting peptide to reach or exceed the predetermined concentration.
  • hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide is also effective in statically inhibiting the binding between hemagglutinin and the influenza virus receptor present on a host cell.
  • the diameter of a peptide aggregate can be measured using, for example, light intensity distribution (intensity PSD).
  • an expression vector capable of expressing such a peptide may be administered.
  • the vector may be a plasmid or a viral vector, but it should have a promoter capable of expressing the inserted gene in a cell to be used.
  • a DNA may be administered in itself; a virus containing this DNA may be infected; and a cell containing this DNA may be transplanted.
  • the virus is not particularly limited but illustratively an adenovirus or a retrovirus, etc. may be used.
  • the vector is constructed so that the virus is capable of infecting cells in the body, and expressing and secreting the hemagglutinin-binding peptide or the influenza virus infection-inhibiting peptide.
  • the vector is constructed so that cells are capable of expressing and secreting the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide.
  • the vector is constructed so that cells express a fusion peptide in which a signal peptide is fused to the N-terminus of the hemagglutinin-binding peptide or influenza virus infection-inhibiting peptide.
  • the method for secretion is not necessarily limited to this technique.
  • filamentous phages fd which present on the out shell surface of peptides having the amino acid sequences of SEQ ID NOs: 1 to 18 were prepared as follows.
  • phage DNAs can be constructed such that a peptide consisting of a 15-residue amino acid sequence encoded by the inserted DNA is expressed on the surface of the phage coat.
  • phages were amplified using the K91Kan host bacteria and phage solution was prepared by a conventional method.
  • hemagglutinin (HA) binding to hemagglutinin (HA) of these phages presenting each of the amino acid sequences of SEQ ID NOs: 2 to 18 on their surface was examined by ELISA.
  • HA hemagglutinin
  • an ether extract of the type A subtype H1 A/PR/8/34 (H1N1) and an ether extract of the type A subtype H3 A/Wuhan/359/95 (H3N2) were used. Specific procedures are described as follows:
  • each well was washed 6 times with 50 mM TBS buffer solution (pH 7.6) and 100 ⁇ l of solution of H1N1 (70 ⁇ l/ml) or solution of H3N2 (70 ⁇ l/ml) was added. After the plate was left for 2 h, the wells were washed 6 times with 50mM TBS buffer solution (pH 7.6), and 100 ⁇ l of 1 mM ethanolamine aqueous solution was added. After the plate was left for 10 min, the wells were washed 6 times with 50 mM TBS buffer solution (pH 7.6) again.
  • A-1 has binding activity comparable to that of the hemagglutinin-binding peptides identified so far ( WO00/59932 and Japanese Laid-Open Application No. 2002-284798 ). (By way of example, the comparison with the binding activity of H3G-1 measured in the same manner is shown in FIG.
  • polypeptides having SEQ ID NOs: 2 to 7, 9 to 10, and 12 to 18 have a higher binding activity to hemagglutinin than the hemagglutinin-binding peptide having the amino acid sequence of SEQ ID NO: 1, and thus useful as a pharmaceutical composition, one of the goals of the present invention.
  • polypeptides having this common sequence are considered to have higher binding activity to hemagglutinin than the hemagglutinin-binding peptide having the amino acid sequence of SEQ ID NO: 1.
  • the peptides having the sequence of s-2 (SEQ ID NO: 3) and the sequence of e-1 (SEQ ID NO: 5), which showed outstandingly high binding to hemagglutinin in the ELISA performed in Example 1, were chemosynthesized (the synthesis was contracted to Toray Industries, Inc.), and their binding to hemagglutinin was evaluated in the form of peptide alone.
  • H3N2 was immobilized on Sensor ChipCM5 (Biacore, BR-1000-14), as with the plate used in Example 1.
  • the binding activity of the peptides to this immobilized H3N2 was evaluated using the Biacore system (Biacore). Specifically, time-course changes of the resonance was measured for each of the peptides at various concentrations.
  • FIG. 4A shows changes in resonance (y-axis) relative to the time (x-axis) course at a peptide concentration of 100 ⁇ M of each hemagglutinin-binding peptide.
  • FIG.4B shows the relative value of the resonance of each hemagglutinin-binding peptide when the amount of binding of A-1 peptide was taken as 1 at a peptide concentration of 200 ⁇ M.
  • Example 1 revealed that the polypeptides having SEQ ID NOs: 2 to 7, 9 to 10, and 12 to 18 had a high binding activity to hemagglutinin, identification of peptides having even fewer residues capable of preventing influenza virus infection was attempted in the following experiments.
  • the peptides shown in the following table 4 were synthesized using the automatic peptide synthesizer PSSM-8 (Shimadzu Co.) by employing Fmoc method. Synthesis reaction of the peptides was performed using Fmoc-SAL-Resin (code A00102, Watanabe Chemical Industries, Ltd.) as solid phase resin. Next, C18-COOH (stearic acid, Sigma) dissolved in dichloromethane was reacted with the solid phase resin after the peptide synthesis to alkylate the N-terminus of these peptides. Then, C18 peptide was excised from the synthesized solid-phase resin to yield C18 peptide amide (with -CONH 2 as its C-terminus).
  • C18 peptide amide was purified by high performance liquid chromatography (HPLC). Further, the molecular weight of the peptide after purification was measured by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For MS measurement, autoflex TM (Bruker Daltonics) was employed using a matrix of ⁇ -cyano-4-hydroxy-cinnamic acis ( ⁇ -CHCA).
  • MDCK Mardin-Darby canine kidney cells were cultured in 6-well microplates as a monolayer in a 5% CO 2 incubator at 37°C until confluence was reached.
  • H1N1 influenza virus solution a solution of type A influenza virus/PR/8/34 diluted to the concentration to form around 100 plaques
  • H3N2 influenza virus solution a solution of type A influenza virus/Aichi/2/68 diluted to the concentration to form around 100 plaques
  • the media in the 6-well microplates was removed and the surface of cells were washed by adding 1 to 2 mL of PBS (-) to each well. After removal of the PBS(-), the samples prepared by the aforementioned method were transferred to the plates (200 ⁇ L/well), which was then incubated at 37°C in a 5% CO 2 incubator for 30 min.
  • C18-s2 (r1-8) was used as the negative control.
  • C18-s2 (r1-8) (SEQ ID NO: 41) is a peptide having the same amino acid composition as s2 (1-8) (SEQ ID NO: 38) but with a different sequence.
  • IC 50 refers to the peptide concentration required to inhibit the infection by 50% when the infection rate without peptides was taken as 100%.
  • C18-s2 (1-8) peptide SEQ ID NO: 38
  • C18-s2 (1-7) peptide SEQ ID NO: 42
  • C18-s2 (1-6) peptide SEQ ID NO: 43
  • C18-s2 (1-5) peptide SEQ ID NO: 44
  • all of which had the ARLPR sequence with the truncated C terminus of SEQ ID NO: 3; as well as C18-s2 (1-4) peptide (SEQ ID NO: 52) were capable of inhibiting H1N1 influenza virus infection more strongly than C18-S2 peptide (SEQ ID NO: 3)) and inhibiting H3N2 influenza virus infection as strongly as, or more strongly than, C18-S2 peptide (SEQ ID NO: 3)).
  • C18-S2 (2-8) peptide was capable of inhibiting H1N1 influenza virus infection as strongly as, or more strongly than, C18-S2 peptide (SEQ ID NO: 3).
  • C18-s2 (1-3) peptide (SEQ IDNO: 54) and C18-s2 (1-2) peptide (SEQ ID NO: 55) were unable to inhibit infection of H1N1 and H3N2 influenza viruses.
  • the above findings revealed that the peptides having the ARLPR sequence with truncated the C-terminus of SEQ ID NO: 3 strongly inhibited influenza virus infection into MDCK cells. It was further revealed that the peptides having the RLPR sequence with N-terminus truncated by one residue strongly inhibited infection of H1N1 influenza virus to MDCK cells. Furthermore, it was revealed that the peptides having the ARLP sequence with truncated C-terminus of SEQ ID NO: 3 also strongly inhibited influenza virus infection into MDCK cells.
  • H1N1 or H3N2 influenza virus infection rate obtained using these peptide by the above-described method is shown in Table 5.
  • C18-s2 (r1-5) peptide (SEQ ID NO: 56) and C18-s2 (r1-4) peptide (SEQ ID NO: 57) were capable of inhibiting infection of H1N1 influenza virus and H3N2 influenza virus into MDCK cells more strongly than C18-s2 (r1-8) (SEQ ID NO: 41).
  • the present invention has made it possible to provide peptides having 4 to 8 residues capable of inhibiting influenza virus infection to a host.
  • the 4- to 8-residue peptide thus obtained can be synthesized at a lower cost than a 15-residue peptide.
  • a peptide having a high affinity for hemagglutinin and a peptide having a high inhibitory activity against influenza virus infection can be provided, and further a pharmaceutical composition using such a peptide having a high affinity for hemagglutinin or a peptide having a high inhibitory activity against influenza virus infection can be provided.

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WO2012018907A3 (en) * 2010-08-03 2012-05-10 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
WO2013138259A2 (en) * 2012-03-13 2013-09-19 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
WO2013121441A3 (en) * 2012-02-14 2013-10-10 Council Of Scientific & Industrial Research Synthetic peptides capable of binding to influenza hemagglutinin protein
US9771395B2 (en) 2014-03-21 2017-09-26 University Of Washington Enhanced influenza hemagglutinin binders

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MY160435A (en) * 2008-06-12 2017-03-15 Univ Putra Malaysia A novel antiviral peptide against avian influenza virus h9n2
WO2010024108A1 (ja) * 2008-08-29 2010-03-04 学校法人慶應義塾 インフルエンザウイルス感染症の予防ないし治療剤
JP5396111B2 (ja) * 2009-03-12 2014-01-22 学校法人慶應義塾 インフルエンザ治療／予防薬
CN102020701B (zh) * 2010-11-12 2012-01-11 重庆大学 一种流感a型h1n1病毒血凝素蛋白亲和肽
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CN102268072B (zh) * 2011-07-06 2014-11-05 中国人民解放军军事医学科学院放射与辐射医学研究所 流感病毒血凝素蛋白结合多肽的结构和用途
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CN103965292B (zh) * 2013-02-01 2020-04-03 中国人民解放军军事医学科学院放射与辐射医学研究所 乙型脑炎病毒包膜蛋白结合肽的结构与用途
US9616114B1 (en) 2014-09-18 2017-04-11 David Gordon Bermudes Modified bacteria having improved pharmacokinetics and tumor colonization enhancing antitumor activity
US10328143B2 (en) * 2014-10-24 2019-06-25 Peptidream Inc. Hemagglutinin-binding peptide
KR101706324B1 (ko) * 2015-01-27 2017-02-14 한국생명공학연구원 인플루엔자 a 바이러스 아형 h1n1 검출용 바이오프로브
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
JP7084627B2 (ja) * 2018-06-06 2022-06-15 株式会社マナＨｓコーポレーション ウイルス感染症予防用組成物

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ229004A (en) * 1988-05-19 1993-09-27 Immunobiology Res Inst Inc Tetrapeptides having t cell helper acitivity
US5104854A (en) * 1989-02-01 1992-04-14 Washington University Antiviral peptides
JP2818761B2 (ja) * 1989-09-14 1998-10-30 財団法人化学及血清療法研究所 非ａ非ｂ型肝炎ウイルス抗原をコードする核酸断片およびその利用法
US6337070B1 (en) * 1993-04-29 2002-01-08 Takara Shuzo Co., Ltd. Polypeptides for use in generating anti-human influenza virus antibodies
WO1995032309A1 (en) * 1994-05-20 1995-11-30 The Mount Sinai School Of Medicine Of The City University Of New York Treatment of influenza virus infection using antivirals that inhibit acylation/palmitylation of hemagglutinin
JP4528989B2 (ja) 1999-03-31 2010-08-25 学校法人慶應義塾 インフルエンザウイルス・ヘマグルチニン結合性ペプチド
CA2941182A1 (en) * 2000-02-16 2001-08-23 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Avirulent, immunogenic flavivirus chimeras
JP4552025B2 (ja) 2000-03-31 2010-09-29 みのる産業株式会社 乗用田植機
JP4651213B2 (ja) 2001-03-27 2011-03-16 株式会社グライコメディクス インフルエンザウイルス・ヘマグルチニン結合性ペプチド
US7442761B2 (en) * 2003-06-06 2008-10-28 Samuel Bogoch Replikin peptides and uses thereof
SE0201863D0 (en) * 2002-06-18 2002-06-18 Cepep Ab Cell penetrating peptides
CN1650012A (zh) * 2002-07-24 2005-08-03 英特塞尔股份公司 来自致病病毒的备选阅读框所编码的抗原
US6924353B2 (en) * 2003-02-27 2005-08-02 Binie V. Lipps Inhibitors for RNA viruses
JP2006101709A (ja) * 2004-09-30 2006-04-20 Glycomedics Inc ヘマグルチニン結合ペプチド、インフルエンザウイルス感染阻害剤、リポソーム、インフルエンザ治療薬、インフルエンザ予防薬
JP2008539209A (ja) 2005-04-26 2008-11-13 カリヨン−シーティーティー リミテッド 診断及び治療剤

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007105565A1 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012018907A3 (en) * 2010-08-03 2012-05-10 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
US8765686B2 (en) 2010-08-03 2014-07-01 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
US9181300B2 (en) 2010-08-03 2015-11-10 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
WO2013121441A3 (en) * 2012-02-14 2013-10-10 Council Of Scientific & Industrial Research Synthetic peptides capable of binding to influenza hemagglutinin protein
CN104220091A (zh) * 2012-02-14 2014-12-17 科学与工业研究会 能结合流感血细胞凝集素蛋白的合成肽
EP2814507A2 (en) * 2012-02-14 2014-12-24 Council of Scientific & Industrial Research Synthetic peptides capable of binding to influenza hemagglutinin protein
US9458196B2 (en) 2012-02-14 2016-10-04 Council Of Scientific & Industrial Research Synthetic peptides capable of binding to influenza hemagglutinin protein
WO2013138259A2 (en) * 2012-03-13 2013-09-19 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
WO2013138259A3 (en) * 2012-03-13 2013-12-19 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
US9388217B2 (en) 2012-03-13 2016-07-12 University Of Washington Through Its Center For Commercialization Polypeptides for treating and/or limiting influenza infection
US9771395B2 (en) 2014-03-21 2017-09-26 University Of Washington Enhanced influenza hemagglutinin binders

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US20100119588A1 (en) 2010-05-13
JPWO2007105565A1 (ja) 2009-07-30
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JP5093100B2 (ja) 2012-12-05
US8299214B2 (en) 2012-10-30
CN101400790A (zh) 2009-04-01

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